Bottom Line:
Cells growing in spheroids responded similarly to the drug, and the inner quiescent fraction also responded after recruitment to the growth fraction.Following normalization against the surviving fraction of cells treated with KPU-300 alone, the surviving fractions of cells irradiated in early M phase coincided.Taken together with potential vascular disrupting function in vivo, we propose a novel radiosensitizing strategy using KPU-300.

ABSTRACTKPU-300 is a novel colchicine-type anti-microtubule agent derived from plinabulin (NPI-2358). We characterized the effects of KPU-300 on cell cycle kinetics and radiosensitization using HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci). Cells treated with 30 nM KPU-300 for 24 h were efficiently synchronized in M phase and contained clearly detectable abnormal Fucci fluorescence. Two-dimensional flow-cytometric analysis revealed a fraction of cells distinct from the normal Fucci fluorescence pattern. Most of these cells were positive for an M phase marker, the phosphorylated form of histone H3. Cells growing in spheroids responded similarly to the drug, and the inner quiescent fraction also responded after recruitment to the growth fraction. When such drug-treated cells were irradiated in monolayer, a remarkable radiosensitization was observed. To determine whether this radiosensitization was truly due to the synchronization in M phase, we compared the radiosensitivity of cells synchronized by KPU-300 treatment and cells in early M phase isolated by a combined method that took advantage of shake-off and the properties of the Fucci system. Following normalization against the surviving fraction of cells treated with KPU-300 alone, the surviving fractions of cells irradiated in early M phase coincided. Taken together with potential vascular disrupting function in vivo, we propose a novel radiosensitizing strategy using KPU-300.

pone.0145995.g003: Characterization of abnormal Fucci fluorescence following KPU-300 treatment.(A) Representative images of abnormal fluorescence after treatment with KPU-300. The time points are shown as hours:minutes in each image; 0:00 represents the start of drug treatment. Bar, 20 μm (B) Relationship between abnormal Fucci fluorescence and M phase following KPU-300 treatment. (a) Two-dimensional flow-cytometric analysis of Fucci fluorescence. The area within a quadrangle represents cells expressing abnormal Fucci fluorescence. (b) Two-dimensional flow-cytometric analysis of DNA content and phosphorylated histone H3 (pHH3). The area within a quadrangle represents cells in M phase. The acquired time points are shown as hours:minutes in each image; 0:00 represents the start of drug treatment. (c) Quantitative analysis of cells with abnormal Fucci expression and those in M phase in Fig 3a and 3b. Data represent means ± S.E. of values obtained from three independent experiments. *p < 0.05; **p < 0.01 vs. controls at time 0.

Mentions:
We previously reported that plinabulin, which has colchicine-like microtubule depolymerization activity, induces abnormal fluorescence in M-phase HeLa-Fucci cells, and that this is an indicator of mitotic catastrophe [39]. In this study, we obtained similar results following treatment with 30 nM KPU-300. During the initial stage of mitosis, only green fluorescence was observed, but abnormal red fluorescence gradually became detectable; subsequently, the cells underwent mitotic catastrophe (Fig 3A). In flow-cytometric analysis, the green fraction gradually shifted upward, becoming double-positive for green and red fluorescence, including most cells after 24 h of treatment (Fig 3Ba). The double-positive fraction was confirmed to be in M phase by immunostaining for phosphorylated histone H3 (Fig 3Bb). A very small fraction of cells showing polyploidy are originally included in HeLa-Fucci cells, which were detected in Fig 3Bb. It is likely that such cells also apparently showed the phosphorylated form of histone H3 after 16 h of treatment time. Quantitative analysis also supported these results (Fig 3Bc)(S4 Table). Considering that the percentage of sub–G1 phase cells (apoptotic fraction) was 16.8% (data not shown) after 24 h KPU-300 treatment, we conclude that almost all cells were arrested in M phase with abnormal fluorescence.

pone.0145995.g003: Characterization of abnormal Fucci fluorescence following KPU-300 treatment.(A) Representative images of abnormal fluorescence after treatment with KPU-300. The time points are shown as hours:minutes in each image; 0:00 represents the start of drug treatment. Bar, 20 μm (B) Relationship between abnormal Fucci fluorescence and M phase following KPU-300 treatment. (a) Two-dimensional flow-cytometric analysis of Fucci fluorescence. The area within a quadrangle represents cells expressing abnormal Fucci fluorescence. (b) Two-dimensional flow-cytometric analysis of DNA content and phosphorylated histone H3 (pHH3). The area within a quadrangle represents cells in M phase. The acquired time points are shown as hours:minutes in each image; 0:00 represents the start of drug treatment. (c) Quantitative analysis of cells with abnormal Fucci expression and those in M phase in Fig 3a and 3b. Data represent means ± S.E. of values obtained from three independent experiments. *p < 0.05; **p < 0.01 vs. controls at time 0.

Mentions:
We previously reported that plinabulin, which has colchicine-like microtubule depolymerization activity, induces abnormal fluorescence in M-phase HeLa-Fucci cells, and that this is an indicator of mitotic catastrophe [39]. In this study, we obtained similar results following treatment with 30 nM KPU-300. During the initial stage of mitosis, only green fluorescence was observed, but abnormal red fluorescence gradually became detectable; subsequently, the cells underwent mitotic catastrophe (Fig 3A). In flow-cytometric analysis, the green fraction gradually shifted upward, becoming double-positive for green and red fluorescence, including most cells after 24 h of treatment (Fig 3Ba). The double-positive fraction was confirmed to be in M phase by immunostaining for phosphorylated histone H3 (Fig 3Bb). A very small fraction of cells showing polyploidy are originally included in HeLa-Fucci cells, which were detected in Fig 3Bb. It is likely that such cells also apparently showed the phosphorylated form of histone H3 after 16 h of treatment time. Quantitative analysis also supported these results (Fig 3Bc)(S4 Table). Considering that the percentage of sub–G1 phase cells (apoptotic fraction) was 16.8% (data not shown) after 24 h KPU-300 treatment, we conclude that almost all cells were arrested in M phase with abnormal fluorescence.

Bottom Line:
Cells growing in spheroids responded similarly to the drug, and the inner quiescent fraction also responded after recruitment to the growth fraction.Following normalization against the surviving fraction of cells treated with KPU-300 alone, the surviving fractions of cells irradiated in early M phase coincided.Taken together with potential vascular disrupting function in vivo, we propose a novel radiosensitizing strategy using KPU-300.

ABSTRACTKPU-300 is a novel colchicine-type anti-microtubule agent derived from plinabulin (NPI-2358). We characterized the effects of KPU-300 on cell cycle kinetics and radiosensitization using HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci). Cells treated with 30 nM KPU-300 for 24 h were efficiently synchronized in M phase and contained clearly detectable abnormal Fucci fluorescence. Two-dimensional flow-cytometric analysis revealed a fraction of cells distinct from the normal Fucci fluorescence pattern. Most of these cells were positive for an M phase marker, the phosphorylated form of histone H3. Cells growing in spheroids responded similarly to the drug, and the inner quiescent fraction also responded after recruitment to the growth fraction. When such drug-treated cells were irradiated in monolayer, a remarkable radiosensitization was observed. To determine whether this radiosensitization was truly due to the synchronization in M phase, we compared the radiosensitivity of cells synchronized by KPU-300 treatment and cells in early M phase isolated by a combined method that took advantage of shake-off and the properties of the Fucci system. Following normalization against the surviving fraction of cells treated with KPU-300 alone, the surviving fractions of cells irradiated in early M phase coincided. Taken together with potential vascular disrupting function in vivo, we propose a novel radiosensitizing strategy using KPU-300.